Metaplastic neuromodulation via transcranial direct current stimulation has no effect on corticospinal excitability and neuromuscular fatigue

Transcranial direct current stimulation (tDCS) is a non-invasive brain stimulation tool with potential for managing neuromuscular fatigue, possibly due to alterations in corticospinal excitability. However, inconsistencies in intra- and inter- individual variability responsiveness to tDCS limit its clinical use. Emerging evidence suggests harnessing homeostatic metaplasticity induced via tDCS may reduce variability and boost its outcomes, yet little is known regarding its influence on neuromuscular fatigue in healthy adults. We explored whether cathodal tDCS (ctDCS) prior to exercise combined with anodal tDCS (atDCS) could augment corticospinal excitability and attenuate neuromuscular fatigue. 15 young healthy adults (6 males, 22 ± 4 years) participated in four pseudo-randomised neuromodulation sessions: sham stimulation prior and during exercise, sham stimulation prior and atDCS during exercise, ctDCS prior and atDCS during exercise, ctDCS prior and sham stimulation during exercise. The exercise constituted an intermittent maximal voluntary contraction (MVC) of the right first dorsal interosseous (FDI) for 10 min. Neuromuscular fatigue was quantified as an attenuation in MVC force, while motor evoked potential (MEP) amplitude provided an assessment of corticospinal excitability. MEP amplitude increased during the fatiguing exercise, whilst across time, force decreased. There were no differences in MEP amplitudes or force between neuromodulation sessions. These outcomes highlight the ambiguity of harnessing metaplasticity to ameliorate neuromuscular fatigue in young healthy individuals. Supplementary Information The online version contains supplementary material available at 10.1007/s00221-024-06874-z.


Introduction
Neuromuscular fatigue is characterised by an exerciseinduced decline in maximal voluntary muscle force (Taylor et al. 2016).It is a prevalent incapacitating symptom recognised as causing decrements in quality of life and disability (Kluger et al. 2013).Both the peripheral and central nervous systems of the body are established to contribute to neuromuscular fatigue (Wan et al. 2017).During neuromuscular fatigue, a gradual decrease in maximal voluntary activation of muscle due to a decline in neural drive occurs centrally (Gandevia 2001).Whilst peripherally, alterations distal or within the neuromuscular junction are apparent (Wan et al. 2017).Prior research has also revealed alterations in the excitability of the corticospinal tract during neuromuscular fatigue (Taylor et al. 1996).In fact, corticospinal excitability is enhanced during single-joint fatiguing exercise, possibly reflecting an increase in motor output to the muscle to counteract the progressive difficulty in maintaining contractile force (Gandevia et al. 1996).
The use of non-invasive brain stimulation techniques such as transcranial direct current stimulation (tDCS) can modify corticospinal excitability (Nitsche and Paulus 2000).tDCS is a portable, non-invasive, painless method of neuromodulation that when employed, delivers continual, low direct electrical current through electrodes on the scalp (Nitsche and Paulus 2000).The effects of tDCS are predominantly a consequence of polarity-specific bidirectional alteration of resting membrane potential (Liebetanz et al. 2002).Anodal tDCS (atDCS) increases cerebral excitability by eliciting subthreshold membrane depolarisation (Nitsche and Paulus 2000).On the other hand, cathodal tDCS (ctDCS) causes a decrease in cerebral excitability and membrane hyperpolarisation (Nitsche and Paulus 2000).Prolonged use of atDCS and ctDCS induces lasting effects similar to persistent forms of activity-dependent synaptic plasticity mediated through strong activation of N-methyl-D-aspartate-type glutamate receptors, namely, long-term potentiation (LTP) and longterm depression (LTD) respectively (Bliss and Lomo 1973;Lüscher and Malenka 2012).
Metaplasticity is referred to regulation of neural activity by which induction of synaptic change is dependent on previous synaptic activity (Abraham and Bear 1996).It operates to preserve synaptic activity within a dynamic range to aid in the integration of temporally dispersed episodes of synaptic change (Abraham and Bear 1996).The Bienenstock-Cooper-Munro theory presents a theoretical explanation on metaplasticity, implying that the threshold for synaptic modification dynamically and bi-directionally varies as a function of prior activity (Bienenstock et al. 1982).Previous LTP activity shifts the modification threshold so that further LTP induction is more difficult, and subsequently increases the likelihood of LTD (Abraham and Tate 1997).The opposite is noticed with previous LTD activity (Abraham and Tate 1997).Prior studies have used two distinct blocks of tDCS over the motor cortex to harness metaplasticity and improve skill acquisition and motor learning in a healthy population (Christova et al. 2015;Fujiyama et al. 2017).For instance, Christova and colleagues found ctDCS preceding atDCS applied concurrently during a grooved pegboard test reduced the completion time of the task when compared to sham condition (Christova et al. 2015).Ultimately, employing priming ctDCS to reduce the modification threshold appears to enhance both corticospinal excitability and functional outcomes of succeeding atDCS (Christova et al. 2015;Fujiyama et al. 2017).Presently, there is a paucity of studies exploring the interaction between metaplastic neuromodulation and neuromuscular fatigue using tDCS.
Currently, no universally efficacious therapy exists for offsetting neuromuscular fatigue (Wan et al. 2017).Given the partial efficacy of existing treatments and the degree to which neuromuscular fatigue can impact an individual's activities of daily living, exploring the relationship between metaplastic neuromodulation and neuromuscular fatigue may be important for uncovering a non-pharmacological and non-invasive treatment approach.Specifically, we explored priming ctDCS prior to single-joint fatiguing exercise combined with atDCS on corticospinal excitability and neuromuscular fatigue in young, healthy adults.We hypothesised that ctDCS primed atDCS applied concurrently with fatiguing exercise would augment corticospinal excitability facilitation and attenuate neuromuscular fatigue compared to atDCS primed by sham stimulation (stDCS).

Participants
Fifteen young healthy volunteers were recruited for the study (6 males, mean age ± SD, 22 ± 4 years).The sample size estimation was performed using G*Power V.3.1.9.4.Due to the design of the study (i.e., repeated measures), it was determined that for an effect size of 0.4 (medium), alpha error of 0.05, power of 0.8, using 95% confidence interval and an expected correlation of the repeated measures of 0.7, 15 participants were required for the study (Cohen 1988).According to the Edinburgh handedness questionnaire, all but one participant were right-handed (handedness Laterality Index, 0.84 ± 0.3), with one individual being ambidextrous (Oldfield 1971).All participants used their right hand during the study sessions.Before participation, volunteers were screened for contraindications to transcranial magnetic stimulation (TMS) (e.g., metallic implants in the skull, cardiac pacemaker, neurological condition, substance abuse, history of seizures, epilepsy, and/or pregnancy).Participants were instructed to refrain from consuming caffeine 4 h prior to the experimental session since ingestion of caffeine has been reported to affect fatigability and performance outcomes (Doherty et al. 2004).The protocol was conducted in accordance with the Declaration of Helsinki and was approved by the Human Research Ethics Committee at the University of Adelaide.The protocol was not registered in a database.Written informed consent was obtained from all participants before involvement.Participants were reimbursed for their time upon completion of the study.

Experimental set-up
Participants sat with their right elbow fixed approximately 90°, pronated forearm aligned on a horizontal surface, and index finger positioned adjacent a force transducer (MLP 100; Transducer Techniques, Temecula, CA).A custom manipulandum was used to restrain the forearm and wrist.
Responses evoked from the right first dorsal interosseous (FDI) muscle were recorded via two electromyography (EMG) surface electrodes (Ag/AgCl) arranged over the muscle in a belly-tendon montage.An additional two grounding electrodes were placed on the wrist and forearm to minimise electrical noise.EMG was amplified (1000x) and band-pass filtered (20 Hz -1 kHz) (1902; Cambridge Electronic Design [CED], UK) prior to digitisation via a 1401 interface at 2 kHz and stored offline.

Experimental protocol
Participants attended the laboratory for four experimental sessions, each separated by at least 7 days to avoid any longterm effects of tDCS (Peters et al. 2013;Reis et al. 2009).
To control for diurnal influence, experimental sessions were completed in the afternoon between the hours of 12 pm and 6 pm (Ridding and Ziemann 2010).During each session, the tDCS paradigm was changed and the order was counterbalanced.The order of these sessions was pseudorandomised and double-blinded with the aid of a separate experimenter, meaning both the participant and main experimenter were blinded to tDCS polarities.Within each of the four experimental sessions, tDCS was applied twice.The initial tDCS functioned to prime subsequent tDCS which was applied during exercise.Priming neuromodulation was either stDCS or ctDCS, whilst stDCS or atDCS was applied during the exercise.Hence, the four experimental sessions were: (1) stDCS-stDCS, (2) stDCS-atDCS, (3) ctDCS-atDCS, (4) ctDCS-stDCS.The stDCS-stDCS and ctDCS-stDCS sessions were control conditions, providing evidence on whether the effects of ctDCS-atDCS were explicitly related to the metaplastic neuromodulation intervention, or merely a result of the fatiguing exercise task.
Before employing tDCS, baseline measures of force and corticospinal excitability were taken (Fig. 1(A).Maximal voluntary contraction (MVC) force was established by calculating the average force of three brief (∼5-second) maximal FDI abduction contractions (no more than 5% variation in force between contractions), each separated by 30-seconds.Baseline corticospinal excitability was measured via eliciting 15 single TMS pulses and 3 peripheral nerve stimulations (PNS).TMS were elicited prior to PNS.Participants then received priming tDCS at rest; ctDCS or stDCS.At 2 min and 8 min post-priming, measurements (15 TMS and 3 PNS) were taken to assess the influence of priming on corticospinal excitability.10 min succeeding the termination of priming, test tDCS stimulation begun; atDCS or stDCS (Fig. 1(B).An inter-stimulation interval of 10 min deemed suitable to ensure test tDCS was executed throughout the after-effects of priming tDCS (Monte-Silva et al. 2010).At the start, middle and end of all tDCS periods, participants reported sensations from the tDCS electrodes.30-seconds following test tDCS commencement, corticospinal excitability measurements (15 TMS and 3 PNS) were repeated.Participants then completed the fatiguing exercise comprising 10 intermittent 30-second MVCs.Between each set (30-seconds), 5 TMS and 1 PNS were elicited.Participants were verbally instructed to start and stop contracting as well as verbally encouraged to perform maximally during the exercise.Force and EMG output were presented on a computer monitor for visual feedback.Post-exercise measurements (15 TMS, 3 PNS followed by 2 brief MVCs separated by 30-seconds) were completed immediately following exercise and repeated 10 min and 20 min after the exercise concluded to gauge neuromuscular fatigue recovery and tDCS after-effects (Fig. 1(C).

tDCS
Constant direct current was delivered by a pair of 35 cm 2 saline-soaked, synthetic sponge electrodes connected to a battery-powered direct current stimulator (NeuroConn DC Stimulator Plus, Germany).A bipolar tDCS electrode montage appears to optimally enhance motor cortex excitability (Nitsche and Paulus 2000) and hence, was used in the current study.The active electrode was centred on the left primary motor cortex (M1), specifically over the representational field of the right FDI muscle.The reference electrode was positioned over the contralateral supraorbital region.tDCS polarity refers to the electrode over the left M1.The real and stDCS, the current was ramped-up and down over 8-seconds at the start and end of the stimulation to inhibit electrical transients.

TMS
TMS was applied to the left M1 to evaluate the excitability of projections from the cortical representation of the right FDI muscle.Single-pulse TMS stimuli were delivered using a standard figure-of-8 magnetic coil (9 cm external wing diameter) connected to a Magstim BiStim unit (MagStim Company, Dyfed, UK).The coil was positioned tangentially M1 hotspot for tDCS was determined as the area of the left hemisphere exhibiting the largest motor evoked potential (MEP) amplitude at 40-60% of maximum TMS stimulator output.The current of the tDCS was set at an intensity of 1 mA (Batsikadze et al. 2013;Jamil et al. 2017;Xian et al. 2023).To replicate previous work, priming ctDCS was applied for 15 min, whilst atDCS during exercise (test tDCS) was administered for 12 min (Fig. 1) (Xian et al. 2023).For stDCS, although the electrode montage was identical to that of genuine tDCS administration, current was only delivered for 30-seconds.Thus, no corticospinal excitability changes were induced during stDCS (Kristiansen et al. 2021).In both ctDCS: cathodal transcranial direct current stimulation.stDCS: sham transcranial direct current stimulation.atDCS: anodal transcranial direct current stimulation.Sec: second.Mins: minutes.TMS: transcranial magnetic stimulation.PNS: peripheral nerve stimulation.5s: five second.30s: 30 second.MVC: maximal voluntary contraction end of contraction) was measured for each of the 30-second MVCs during the exercise.The root mean squared EMG was measured for every 5% maximum EMG contraction to ensure all measurements were taken at a constant EMG level.
Peak-to-Peak MEP and Mmax amplitudes were calculated in millivolts using offline recordings on Spike2 software.MEP amplitudes were measured between 20 and 50 milliseconds after the TMS pulse, whilst Mmax amplitudes were measured between 3 and 30 milliseconds following PNS.MEP amplitude at individual time points were determined as the mean amplitude across all trials in the measurement block.MEPs were normalised to Mmax (MEP % Mmax) to study corticospinal changes whilst excluding changes at the level of the muscle.

Statistical analysis
IBM SPSS Statistics software (Version 24; Chicago, USA) was used for the statistical analyses.Separate linear mixed model (LMM) analyses with factors time and neuromodulation (stDCS-stDCS vs. stDCS-atDCS vs. ctDCS-atDCS vs. ctDCS-stDCS) were used to assess main effects and interactions.For MEP, LMM analyses were performed for baseline and post-priming, 30-seconds post-tDCS and all the fatiguing contraction time points, and baseline and post-exercise.Furthermore, two LMM analyses were completed for force: one for all the fatiguing contraction time points and another for baseline and post-exercise.For all comparisons, normality of the data was validated using Shapiro-Wilk tests.Post hoc tests with Bonferroni's correction for multiple comparisons were employed to probe significant main and interaction effects.One-way analyses of variance were used for evaluating differences between neuromodulation conditions in time of day, lab temperature and humidity.All data in text and tables are conveyed as mean ± SD.Data in figures are illustrated as box-and-whisker plots, with the "box" depicting the median and the 25th and 75th quartiles, and the "whisker" highlighting the 5th and 95th percentile.The value of alpha was set at ≤ 0.05.

Results
Participant characteristics are displayed in Table 1.No adverse reactions to tDCS were reported by participants.Participants were unable to distinguish stDCS from real tDCS since sensations related with real stimulation were also communicated during stDCS.The sensations reported comprised needling, warmth, burning, stinging, prickling, itchiness, and tingling.Most described sensations at the beginning of tDCS but, felt nothing when asked in the over the brain at a 45° angle to the sagittal plane to deliver a posterior-anterior current flow, and to optimally elicit MEPs in the FDI.The amplitude of MEP was used to assess corticospinal excitability.All TMS pulses were delivered while participants contracted their right FDI muscle at 5% of their maximum EMG (as determined during baseline MVC trials).An active state was chosen for practicality since a lower stimulation intensity is required to consistently produce a MEP if muscle is active (Ngomo et al. 2012).Additionally, and perhaps more importantly, active muscle more closely represents what occurs during a task (i.e., fatiguing exercise).The FDI motor hotspot was established by mapping (at 40-60% of maximum stimulator output) for the area that resembled the largest MEP amplitude.The position of the coil was marked on the tDCS electrodes and scalp with permanent marker to aid in consistent coil positioning.These markings were continuously monitored throughout the protocol.Active motor threshold (AMT) was defined as the lowest TMS stimulus intensity necessary to elicit a MEP discernible from background EMG signal in 3 out of 5 trials (Sidhu et al. 2017).TMS intensity was set at 120% of AMT for all measurement blocks (stDCS-stDCS: 56 ± 9%; stDCS-atDCS: 55 ± 11%; ctDCS-atDCS: 55 ± 10%; ctDCS-stDCS: 54 ± 8%; of maximum stimulator output, One-way ANOVA main effect of session: P = 0.89) to ensure effective activation of the FDI during the experiment.

PNS
The right ulnar nerve was peripherally stimulated via a bipolar bar electrode probe connected to a constant-current stimulator (DS7A; Digitimer, Hertfordshire, UK).Upon establishing the optimal position, the probe was secured so that the cathode was angled distally.The optimal position was established as the site eliciting the greatest compound muscle action potential (M-wave) in resting FDI at 10 mA current.To ascertain the maximum M-wave (Mmax), stimulation intensity was incrementally increased by 5 mA until the M-wave amplitude ceased to rise.Stimuli were delivered at a test intensity of 120% of the intensity necessary to produce Mmax (stDCS-stDCS: 27 ± 6 mA; stDCS-atDCS: 26 ± 6 mA; ctDCS-atDCS: 28 ± 7 mA; ctDCS-stDCS: 28 ± 6 mA; One-way ANOVA main effect of session: P = 0.50).

Main findings
The current study is the first to explore the interaction between metaplastic neuromodulation and neuromuscular fatigue in young, healthy individuals.Data on tDCSinduced modulation of MEP amplitudes in young healthy participants showed no significant shift in corticospinal excitability with ctDCS-atDCS compared to stDCS-atDCS, no differences in MEP amplitudes between the explored neuromodulation conditions.Hence, our hypothesis of an augmentation in corticospinal excitability facilitation with ctDCS primed atDCS applied simultaneously with fatiguing exercise is not confirmed in this study.
Maximal compound muscle action potential, or Mmax, signifies full activation of the motor neurone pool of a muscle (Magladery et al. 1951).Studies reveal a decrease in Mmax amplitude in the presence of muscular fatigue (Crone et al. 1999).In the present study, it is likely that to some degree, the attenuation in Mmax is a result of the exercise protocol inducing fatigue.At a physiological level, an impairment of neuromuscular propagation is a probable cause for this attenuated Mmax (Fuglevand et al. 1993).However, there were several instances across several participants (6 data points out of a total of 600 in the data set: 1% of data) whereby the MEP was greater than Mmax.Since it is not physiologically possible for TMS to markedly recruit more motoneurons than supramaximal stimulation of motoneuron

tDCS effects on corticospinal excitability
Our current understanding of an enhancement of MEP amplitudes during fatiguing maximal efforts (i.e.MVCs) is attributed to increases in corticospinal excitability (Taylor et al. 1996), arising as a counteractive mechanism to boost contractile force as neuromuscular fatigue develops (Gandevia et al. 1996).The notion that corticospinal excitability increases during single-joint fatiguing exercise (Benwell et al. 2007;Otieno et al. 2019) is verified in the present study by an enhancement of MEP amplitudes in the stDCS-stDCS condition.The Bienenstock-Cooper-Munro theory states that the threshold for synaptic modification dynamically and bi-directionally adjusts as a function of former activity (Bienenstock et al. 1982).Although it is understood that the induction of excitability modification is sensitive to the state of the network enforced by prior synaptic activity (Abraham and Bear 1996;Abraham and Tate 1997;Bienenstock et al. 1982;Siebner et al. 2004;Lang et al. 2004), we found Fig. 3 Mean MVC force during-(A) and post-(B) fatiguing exercise displayed over time and across neuromodulation conditions (stDCS-stDCS, stDCS-atDCS, ctDCS-atDCS, ctDCS-stDCS) for fifteen young healthy adults.Bonferroni's post hoc tests: * indicates significant difference from fatiguing contraction 1 (Fatigue 1) (P < 0.05); # denotes significant difference from baseline (P < 0.05).Data are represented as box-and-whisker plots, with the "box" depicting the median and the 25th and 75th quartiles, and the "whisker" highlighting the 5th and 95th percentile.tDCS: transcranial direct current stimulation.stDCS: sham tDCS.atDCS: anodal tDCS.ctDCS: cathodal tDCS.stDCS-stDCS: stDCS applied during priming and exercise.stDCS-atDCS: stDCS applied during priming and atDCS applied during exercise.ctDCS-atDCS: ctDCS applied during priming and atDCS applied during exercise.ctDCS-stDCS: ctDCS applied during priming and stDCS applied during exercise.N: newton.Min: minute Xian et al. 2023).In fact, it has been observed that the aftereffects of neuromodulation are variable within and between participants in relation to direction, duration, and magnitude (Huang et al. 2017) and influenced by electrode and skull characteristics (Antonenko et al. 2021).A variety of factors may play a role in the neuromodulation response variability including sex, attention, physical activity levels and optimal stimulation dose (Fertonani and Miniussi 2017;Guerra et al. 2020;Huang et al. 2017;Li et al. 2015;López-Alonso et al. 2014;Ridding and Ziemann 2010;Rudroff et al. 2020).Hence, future studies exploring the relationship between corticospinal excitability and different tDCS parameters are necessary to decipher the most appropriate arrangement for enhancing tDCS outcomes (Amann et al. 2022).

tDCS effects on neuromuscular fatigue
The ability of neuromodulation to mutually modify motor performance and corticospinal excitability highlights the possibility of exploiting neuromodulation to influence mechanisms of neuromuscular fatigue (Christova et al. 2015;Fujiyama et al. 2017).The present study reveals an attenuation of MVC force during exercise regardless of the neuromodulation condition.MVC remained significantly lower than baseline across all four conditions at 20 min post-exercise.Kan et al. (2013) reported similar results in their study, wherein atDCS had no effect on MVC strength or time-to-task-failure (TTF) of the elbow flexors (Kan et al. 2013).However, unlike the delivery of tDCS during exercise in the present study, Kan et al. (2013) applied atDCS prior to exercise.Conversely, Williams et al. (2013) reached differing conclusions in their study, whereby application of atDCS during fatiguing exercise led to an improvement in performance.A fundamental distinction in the protocol that could justify the contradictory result concerns the quantification of neuromuscular fatigue.Williams et al. (2013) assessed neuromuscular fatigue by TTF in submaximal isometric contractions (as opposed to intermittent MVCs in the current study).Consequently, it is probable that amelioration of neuromuscular fatigue via tDCS is task dependent and regulated, at least partially, by exercise intensity.
Since development of neuromuscular fatigue is related to an increase in corticospinal excitability (Benwell et al. 2007;Otieno et al. 2019), a parallel between fatigability and excitability of outcomes is expected.Earlier research has demonstrated an attenuation of neuromuscular fatigue upon delivery of atDCS concurrently with exercise (Oki et al. 2016;Williams et al. 2013).Yet, investigations on priming ctDCS prior to concurrent application of atDCS during a task have illustrated enhanced corticospinal excitability and motor performance when compared to atDCS with no priming (stDCS) (Christova et al. 2015;Fujiyama et al. 2017).
axons, we suspect that there were methodological issues in obtaining Mmax (e.g.movement of the stimulation probe from the optimal position on the nerve) during the fatiguing exercise task.In any case, analysis of MEP (unnormalised) resulted in similar outcomes compared to MEP (normalised to Mmax) -suggesting that the MEP outcome as reported in the current study stands independent of muscle changes.Interestingly, in comparison to stDCS-stDCS neuromodulation, Mmax at the beginning of the fatiguing exercise (30-seconds post-test tDCS) was greater in stDCS-atDCS and ctDCS-stDCS conditions.The underlying reason for the differences in Mmax between sessions is not clear and may require further investigation in future work.
Notably, there was a lack of difference in corticospinal excitability between stDCS-atDCS and stDCS-stDCS neuromodulation in this study that conflicts with previous investigations exploring physiological and behavioural outcomes of tDCS (Cogiamanian et al. 2007;Kidgell et al. 2013;Nitsche andPaulus 2000, 2001;Nitsche et al. 2002;Williams et al. 2013).Earlier studies examining the application of atDCS during muscle activity have revealed increases in corticospinal excitability (Hendy and Kidgell 2013;Kim and Ko 2013).The lack of difference between stDCS-atDCS and stDCS-stDCS in the current study compared to similar research in the field (Cogiamanian et al. 2007;Williams et al. 2013) may be due to variation in methodologies and study designs.For example, unlike the present study, many tDCS investigations have failed to blind the experimenter involved in the research (Cappa 2008;Marangolo et al. 2011;Monti et al. 2008;Norise et al. 2017;Shah-Basak et al. 2015); increasing the risk of bias.Additionally, the experimental parameters of tDCS vary among studies.Factors such as timing, intensity, and duration of tDCS application are known to influence tDCS outcomes (Sellaro et al. 2016).In this study, tDCS was applied at an intensity of 1 mA yet, other studies in the area commonly employ a tDCS intensity of 1.5-2 mA (Abdelmoula et al. 2019;Cogiamanian et al. 2007;Fujiyama et al. 2017;Kan et al. 2013).It has been established however, that higher tDCS intensities does not necessarily increase corticospinal excitability if more than 1.5 mA is administered (Kan et al. 2013).Likewise, the duration of tDCS application in this study was 15 min of priming and 12 min during the exercise task but, studies reporting an effect of stDCS-atDCS apply tDCS during performance of a motor task for 20 min (Christova et al. 2015;Fujiyama et al. 2017).Batsikadze and colleagues have demonstrated that an increase in tDCS intensity or duration is not strictly associated with an enhancement of its efficacy but may in fact alter the direction of its effects (Batsikadze et al. 2013).Inter-individual variability of response to tDCS may also contribute to the conflicting outcomes of the present study to that of prior investigations (Chew et al. 2015; is some indication of increased variability if fewer than 20 simultaneous MEP responses are averaged (Biabani et al. 2018).Thus, the 5 TMS-evoked MEP responses during fatiguing exercise, and 15 at every other time point, should be acknowledged.

Conclusion
This work offers a novel application of tDCS to modulate corticospinal excitability and fatigability in young, healthy adults.In contrast to prior investigations, we observed no differential modulation of corticospinal excitability or neuromuscular fatigue with delivery of atDCS during isometric single-joint fatiguing exercise.Consequently, priming ctDCS prior to fatiguing exercise combined with atDCS failed to augment corticospinal excitability facilitation or attenuate neuromuscular fatigue.Overall, the present results highlight some limitations in the use of tDCS to attenuate muscle fatigability.Further exploration to establish the efficacy of tDCS and the underlying neurophysiological mechanisms in mitigating neuromuscular fatigue in both normal and pathological settings is warranted.

Corticospinal Excitability
The

Limitations
The current study has some considerations that should be addressed.Firstly, it could be disputed that capitalisation of metaplasticity was not effective as the priming alone caused no modification in MEP.Yet, even when priming stimulation fails to produce noticeable variations in excit-

Table 1
Demographic characteristics of participants.Work, sport, and leisure time indices are sub-scales of the International Physical Activity Questionnaire(Baecke et al. 1982).Participant scores ranged from 1 (sedentary) to 5 (active) for each index.cm: centimetre.kg: kilogram ctDCS-stDCS or excitability facilitation induced by the fatiguing exercise itself (stDCS-stDCS).Likewise, there was no benefit of tDCS, either primed or not primed, for the development of neuromuscular fatigue during exercise.Since this study revealed no significant benefit of ctDCS-atDCS on enhancing corticospinal excitability or reducing neuromuscular fatigue during exercise, the prospect of harnessing metaplasticity to ameliorate neuromuscular fatigue in young healthy individuals remains elusive.
(Herwig et al. 2001;sHunter 2014)rations have been shown to occur(Abraham 2008; Abraham and Bear 1996; Sidhu  2021).Moreover, several variables are well-established in influencing the outcomes of tDCS.Although we are aware of hormones and the potent regulatory role they play in plasticity(Abraham 2008; Ansdell et al. 2019), we did not control for the menstrual cycle in female participants and is considered a limitation.Additionally, sex differences in fatigability are documented(Ansdell et al. 2020;Hunter 2014).Females are commonly described as having greater resistance to neuromuscular fatigue because of greater availability of oxygen during exercise(Ansdell et al. 2019(Ansdell et al.  ,  2020)).Regarding study techniques, neuronavigation was not employed in our study.Neuronavigation systems allow for accurate identification of the brain area of interest by utilisation of an individual's magnetic resonance imaging (MRI) data(Sparing et al. 2010).With the absence of MRI, neuronavigation systems may be paired with anatomical data and optically tracked frameless stereotaxic systems to allow for continual tracking of a hotspot(Orringer et al.  2012).While neuronavigation is common in TMS studies for precise positioning of the magnetic coil, it is of greater importance in studies exploring brain regions other than M1 (e.g., frontal cortex)(Herwig et al. 2001; Sparing et al.  2010).A further limitation of the present work is the fact that the effectiveness of neuromodulation (i.e., tDCS) and neuromuscular fatigue appear to be muscle and task dependent(Otieno et al. 2022; Simione et al. 2018).Hence, the outcomes of the current study in the FDI may not necessarily be extrapolated to muscle groups that are commonly used during activities of daily living and with a different topographical representation in M1.It could also be argued that the relatively homogenous young healthy study sample is a limitation.However, investigating the basic neurophysiological mechanisms of neuromuscular fatigue in a healthy cohort has broader significance for the field.Lastly, there